Enantiomers

Alanine, one of the amino acids in your proteins, comes in two forms that are mirror images of each other. Your ribosomes build proteins from only one of them. Bacteria use the other one in their cell walls. Yet in a flask the two forms melt at the same temperature, dissolve equally well and give identical IR and NMR spectra. Only two kinds of test can tell them apart: polarized light, and another molecule that is itself chiral.

The definition

Look at the two alanine molecules below. Each central carbon carries the same four groups: NH₂, COOH, CH₃ and H. The right-hand drawing is the reflection of the left-hand one in the dashed mirror. However you turn either one, it will not sit exactly on the other, so they are two different molecules.

Two stereoisomers that are mirror images and cannot be superimposed are called enantiomers. (Stereoisomers have the same atoms joined in the same order and differ only in how those atoms are arranged in space.) Chirality looked at one molecule and its reflection; this page treats the two as a pair of compounds.

Apply the rules from R/S configuration: the left molecule is (S)-alanine and the right one is (R)-alanine. That is no accident. A reflection reverses the direction in which you trace priorities 1, 2 and 3, so it turns every R into S and every S into R.

The same holds with more than one stereocenter. Take (2R,3R)-3-bromobutan-2-ol. Its mirror image inverts both centers, so its enantiomer is (2S,3S). Invert only C3 instead and you get (2R,3S). That molecule is not a mirror image of the first, so it is not an enantiomer. It is a diastereomer: a stereoisomer that is not a mirror image. Diastereomers covers that relationship.

Every chiral molecule has exactly one enantiomer: the molecule with every stereocenter inverted. Some molecules have stereocenters yet are superimposable on their mirror image. Inverting all their centers gives back the same molecule, so they have no enantiomer. Meso compounds covers these.

Identical physical properties, with one exception

Put (S)- and (R)-alanine through the usual lab tests and nearly every result matches. The table lists the tests that agree first, then the two that do not.

The matching rows are not a weakness of the instruments. Every bond length, bond angle and energy in one enantiomer is exactly matched in the other. A measurement that depends only on those, or on how the molecule meets something achiral such as water, has to give the same answer for both.

Both rows that differ involve something chiral. Receptors come later on this page; the other row is optical activity: a sample that holds more of one enantiomer than the other turns the plane of polarized light.

Ordinary light vibrates in every plane at once, and plane-polarized light has been filtered so that it vibrates in only one. That beam can be treated as two beams whose vibration twists as they travel, one to the left and one to the right. A chiral sample slows one of them slightly more than the other, and the plane comes out turned.

Optical activity was known before anyone could draw a molecule in three dimensions. In 1848 Pasteur found that two kinds of crystal of a tartrate salt gave solutions that turned polarized light in opposite directions. He argued that the molecules themselves must be mirror images.

Specific rotation

The measurement is made in a polarimeter. A polarizing filter lets through light in one plane only. The light then crosses a tube of the sample, and a second filter, the analyzer, is turned until the light comes through again. The angle it had to be turned through is the observed rotation, α, in degrees.

The observed rotation depends on how many molecules the light meets on its way through, so it changes with the tube length and the concentration. Dividing both out gives the specific rotation, [α], a number that belongs to the compound itself:

Here α is the observed rotation in degrees, l is the path length in decimeters (a standard tube is 1 dm long) and c is the concentration in g/mL. For a pure liquid with no solvent, c is its density. Specific rotation also changes with temperature, with the color of the light and, often a great deal, with the solvent, so it is quoted with all three. (S)-Alanine is +14.5° in 6 M hydrochloric acid but only about +2 to +3° in water, and some compounds even change sign. The usual light is the yellow sodium D line (589 nm), and [α]_D²⁰ means that light at 20 °C.

A label can carry the configuration and the sign together. (S)-(+)-Carvone, which smells of caraway, is +61°; (R)-(−)-carvone, which smells of spearmint, is −61°.

Racemic mixtures

Start with a flat carbocation: a carbon with only three bonds and a positive charge, the kind that forms when 2-bromobutane loses its bromide. (You meet it properly in How Reactions Happen.) The positive carbon and its three neighbors lie in one plane, so the carbon has two identical faces. A bromide ion can bond to either face with equal ease.

Bonding from above gives (R)-2-bromobutane, and bonding from below gives (S)-2-bromobutane. The two form in equal amounts. A 50:50 mixture of two enantiomers is a racemic mixture, or racemate, written (±). Its rotation is zero, because the two enantiomers cancel each other. Turning a single enantiomer into a racemate is called racemization.

A racemate forms whenever a new stereocenter is made and nothing in the flask favors one mirror image. That means achiral starting materials, achiral reagents, an achiral catalyst (or none) and an achiral solvent. A flat radical behaves the same way as the flat cation, which is why radical halogenation (a preview of How Reactions Happen) gives racemic products. Make any one of them a single enantiomer, such as a chiral catalyst, and the two products no longer have to form equally.

Real reactions through a carbocation (the SN1 reaction in Substitution & Elimination) often fall short of 50:50. The leaving group can stay beside the face it left for a moment and partly block it, so the product often carries some excess of the inverted enantiomer (commonly 55:45 up to about 70:30, as SN1 shows). Most of the starting configuration is still lost, and that loss is what marks the mechanism. SN2, the other substitution in that chapter, inverts every molecule.

As solids, racemates come in two kinds. Most crystallize as a racemic compound: each crystal holds (R) and (S) molecules side by side in a 1:1 pattern. It is a distinct solid, and its melting point can be higher or lower than the pure enantiomer’s. A minority crystallize as a conglomerate: a mixture of separate crystals, each made of only (R) or only (S) molecules. Like a mixture of two different solids, a conglomerate melts lower than either pure enantiomer.

Pasteur’s tartrate salt happened to be a conglomerate, and its two kinds of crystal had mirror-image shapes. That is the only case in which you can separate enantiomers by picking crystals apart, and he did it with tweezers.

Enantiomeric excess

Suppose a sample is 90% (S) and 10% (R). Pair each (R) molecule with one (S) molecule: those pairs make up 20% of the sample, and they are a racemate that contributes no rotation. The remaining 80% is (S) with no partner. That leftover is the enantiomeric excess, ee.

Only the excess turns the light, so a polarimeter can measure ee. The measured rotation as a percentage of the pure enantiomer’s rotation is called the optical purity:

The two numbers agree when each molecule turns the light by the same amount whatever it is mixed with. This holds for almost every compound. In a few, the molecules pair up in solution and the two numbers drift apart, so careful work measures ee directly by chiral chromatography, described below.

To go back from ee to composition, split the racemic part evenly between the two enantiomers. The major enantiomer is (100 + ee) ÷ 2 percent of the sample, and the minor one is the rest.

A racemate has ee = 0%, and a single pure enantiomer has ee = 100%. Asymmetric synthesis, a reaction built to make more of one enantiomer than the other, usually with a chiral catalyst, is judged by the ee it delivers.

Why they behave differently in biology

An enzyme or a receptor is built from chiral amino acids, so its binding site is chiral too. Picture a site with three pockets, one each for the NH₂, COOH and CH₃ of alanine. (S)-alanine can put all three groups in their own pockets. Its mirror image cannot, however it is turned, in the same way that a left hand will not fit a right-handed glove.

The two complexes, (S)-alanine on the receptor and (R)-alanine on the same receptor, are not mirror images of each other. The mirror image of the first would need a mirror-image receptor, and your body does not make one. The two complexes are therefore diastereomers (see The definition above), and diastereomers have different energies. So the two enantiomers bind with different strengths. The same idea explains why a drug’s enantiomers can act so differently, with examples in Chirality.

Separating enantiomers

Separating the two enantiomers of a racemate is called resolution. Distillation, ordinary recrystallization and ordinary chromatography cannot do it, because they rely on the properties that enantiomers share. The fix is to bring in something chiral.

Ibuprofen, a carboxylic acid, is made as a racemate. In the lab it can be resolved with (S)-1-phenylethylamine, a chiral amine available as a single enantiomer. The amine is a base, so it turns each ibuprofen molecule into a salt. (R)-ibuprofen gives the (R)-acid·(S)-amine salt, and (S)-ibuprofen gives the (S)-acid·(S)-amine salt.

The two salts have opposite configurations at the acid and the same one at the amine. They are not mirror images, so they are diastereomers, and their solubilities are different. The less soluble salt crystallizes and is filtered off. Adding aqueous HCl to it gives back the free carboxylic acid, highly enriched in one enantiomer, while the amine stays in the water as its hydrochloride salt and can be recovered. The salt left in solution gives ibuprofen rich in the other enantiomer. Recrystallizing the salt until its rotation stops changing, before the HCl step, makes the acid essentially pure.

The resolving agent must be a single enantiomer. Chemists often use a cheap natural one, such as tartaric acid or brucine, or a simple chiral amine like this one. A racemic amine would give four salts in two mirror-image pairs, and each pair would share every property again.

Chiral chromatography uses the same idea without making salts. Its column is packed with a solid that carries a single enantiomer of some chiral group. The two enantiomers of the sample stick to it with different strengths, so they come off the column at different times. In the lab today, chiral chromatography is the more common route, both for separating enantiomers and for measuring ee.

Resolution has a built-in cost. At most half of a racemate is the enantiomer you want, unless the other half can be racemized and fed back in. That is why making a single enantiomer directly, by asymmetric synthesis, is so valuable.

What carries forward

Enantiomers are one of the two kinds of stereoisomer relationship; Diastereomers covers the other. When a single enantiomer goes in and a racemate comes out, the reaction usually passed through a flat intermediate: the carbocation of SN1 in Substitution & Elimination or, in the same way, the flat radical of How Reactions Happen. Laboratory syntheses of amino acids give racemates that must then be resolved, as Biomolecules shows. And using a chiral partner to turn enantiomers into diastereomers underlies every chiral separation and every asymmetric reaction.